The principle of solid phase oligonucleotide synthesis traces its history to work of Merrifield, Khorana and others in the 1950's and 1960's. The development of automated synthetic methods over the past decade has had a major impact in the fields of molecular biology and biological chemistry.
The stepwise synthesis of deoxyoligonucleotides generally involves the formation of successive phosphodiester bonds between 5'-hydroxyl groups of bound nucleotide derivatives and the 3'-hydroxyl groups of a succession of free nucleotide derivatives.
The synthetic process typically begins with the attachment of a nucleoside derivative at its 3'-terminus by means of a linker arm to a solid support, such as silica gel or beads of borosilicate glass (also known as controlled pore glass, "CPG") packed in a column.
The ability to activate one group of the free nucleotide derivative requires that other potentially active groups elsewhere in the reaction mixture be "protected" by reversible chemical modification. The reactive nucleotide derivative is a free monomer in which the 3'-phosphate group has been substituted, e.g., by dialkylphosphoamidite, which upon activation reacts with the free 5'-hydroxyl group of the bound nucleoside or oligonucleotide to yield a phosphite triester. The phosphite triester is then oxidized to a stable phosphotriester before the next synthetic step.
The 3'-hydroxyl of the immobilized reactant is protected by virtue of its attachment to the support, and the 5'-hydroxyl of the free monomer can be protected by a dimethoxytrityl ("DMT") group in order to prevent self-polymerization.
Additionally, the reactive groups on the individual bases are also protected. A variety of chemistries have been developed for the protection of the nucleoside exocyclic amino groups. The use of N-acyl protecting groups to prepare N-acylated deoxynucleosides has found wide acceptance for such purposes.
After each reaction excess reagents are washed off the column, any nonreacted 5'-hydroxyl groups are blocked or "capped" using acetic anhydride, and the 5'-DMT group is removed using either 80% acetic acid, or dichloro- or trichloro-acetic acid in dichloromethane, to allow the extended bound oligomer to react with another activated monomer in the next round of synthesis.
Synthetic methodologies that were in common use only a decade ago, such as the phosphodiester method, are now largely obsolete. Today almost all synthetic oligonucleotides are prepared by solid phase phosphoramidite techniques. See generally T. Brown and D. Brown, "Modem machine-aided Methods of Oligonucleotide Synthesis", Chapter 1, pp. 1-24 in Oligonucleotides and Analogues, A Practical Approach, F. Eckstein, ed., IRL Press (1991); and H. Weber and H. G. Khorana, J. Mol. Biol., 72: 219-249 (1972).
Upon completion of synthesis, the solid phase bound oligomer is removed from the support (cleaved) and deprotected in order to remove all remaining protecting groups from the oligonucleotide. Typically, the cleavage is performed at room temperature using concentrated ammonium hydroxide (30% by volume) while the solid support is kept in the synthesis column. The amount of ammonium hydroxide used for the cleavage generally ranges from 1 ml to 3 ml. The ammonium hydroxide can be delivered to one end of the column through one syringe and collected at the other end in another syringe. The feeding of ammonium hydroxide through the column is repeated several time during the cleavage process, which typically is performed for 1-2 hours (see, e.g., protocol 5 of Brown and Brown, cited above, as well as Schulhoff, et al., Nucleic Acids Research, 15: 397-416 (1987)
After completion of the cleavage of the oligomers from the support, the remaining protecting groups on the nucleotide building blocks are removed by incubation in an ammonium hydroxide solution. The incubation is typically carried out either at room temperature for 24 hours, or with heating, e.g., by heating to 55.degree.-60.degree. C. for 6-17 hours; to 70.degree. C. for about 3 hours; or to 85.degree. C. for 30-60 minutes. The heating of ammonium hydroxide, especially above 55.degree. C. is potentially dangerous however, due to build-up of pressure within the tube or vial.
The cleavage/deprotection can also be performed in a separate vial if the column is opened up and the contents emptied into the vial. Ammonium hydroxide is added, the vial is sealed, and the contents are then incubated with ammonium hydroxide as described above. Drawbacks associated with this method include the additional labor required to open the column, together with the corresponding potential loss or solubilization of support material, as well as the need to separate the support from the ammonium hydroxide prior to evaporation.
The particular cleavage and deprotection protocol used in any situation is largely determined by the chemistry of the protecting groups used for the DNA building blocks. The use of some protecting groups dates back 25 years, to the early work of Schaller et al., J. Amer. Chem. Soc. 85, 3821-3827 (1963), Buchi and Khorana, J. Mol. Biol. 72, 251 (1972)). These groups include benzoyl(bz) and isobutyryl(i-bu) protecting groups of adenosine A(bz), cytosine C(bz) and guanosine G(i-bu). Generally, thymidine T is not protected.
More labile protecting groups of the phenoxyacetyl type have recently been introduced commercially (e.g., Expedite.TM. and "PAC" amidites introduced by Millipore and Pharmacia, respectively). These types of protecting groups have been known for some time and their stability as monomer nucleosides have been studied. See, for instance, Koster et al., Tetrahedron, 37: 363-369 (1981) which describes the use of a 1:1 methanol:NaOH mixture as a deacylating agent to determine the stability of various N-protecting groups.
Their use in DNA synthesis has also been investigated (Schulhoff, et al., Nucleic Acids Research, 15: 397-416 (1987), Schulhoff, et al. Nucleic Acids Research., 16: 319 (1988), and Sinha et al., Biochimie, 75: 13-23 (1993)). The conventional protecting groups A(bz), C(bz) and G(i-bu), however, will likely continue to dominate the DNA synthesis field for a long time since it appears that they tend to be more stable in storage and use.
The increased reactivity of the new, more labile, protecting groups also require precautions during synthesis. The capping reagent acetic anhydride/pyridine/methylimidazole is typically replaced with a capping reagent that provides the same function as the protecting groups (e.g., t-butylphenoxyacetic anhydride instead of acetic anhydride), thereby providing for an easy exchange of such groups on the nucleosides.
According to instructions provided by the manufacturer, DNA made using the Expedite.TM. amidites can be deprotected by incubation for 15 minutes at 55.degree. C. or 2 hours at room temperature. If the DNA is first cleaved from the solid support (using concentrated Ammonium hydroxide) while still in the column the cleavage time is set at 90 minutes at room temperature. If the support is instead emptied into a vial containing ammonium hydroxide, the combined cleavage and deprotection time is 15 minutes at 55.degree. C., or 2 hours at room temperature. (See "Instructions for using Expedite Chemistry", Millipore).
In yet another approach, Beckman company has recently introduced a "One-Hour DNA" technology involving the use of what are described as "acetyl-protected deoxyC phosphoamidites". Beckman describes the cleavage and deprotection as taking ten minutes, followed by drying of an aliquot of the sample within another ten minutes. The technology appears to be limited in that it appears to again be an ammonia-based method that relies on the volatility of the ammonium hydroxide for the rapid drying time. It also appears to require the use of a dedicated "synthesizer" instrument, that itself is of limited capacity as well as new amidite chemistry.
Sodium hydroxide has found limited use for the cleavage or deprotection of modified DNA, but does not appear to have been previously used or described for use in the preparation of synthetic oligonucleotides. For instance, Dreyer and Dervan, Proc. Nat'l. Acad. Sci., 82: 968-972 (1985) describe the use of 0.1 molar sodium hydroxide to cleave oligomer from a support for 6 hours at room temperature. Griffin and Dervan, J. Am. Chem. Soc., 114: 7976-7982 (1992) describe a sodium hydroxide deprotection time of 20 hours wherein the recovery procedures is estimated to take 2-3 days. It is of interest to note that the authors in each case used the traditional ammonium hydroxide cleavage/deprotection protocol for non-modified DNA (i.e., naturally occurring DNA). The methodology used in the sodium hydroxide procedure differs significantly from the traditional ammonium hydroxide protocol, and the overall sodium hydroxide-based protocol still required on the order of days.
Following cleavage and deprotection, synthesized DNA is typically recovered by evaporation. Evaporation of 1-3 ml of ammonium hydroxide on a standard laboratory evaporator (Speedvac.RTM., Savant) takes between 1.5-3 hours. If the DNA is fully deprotected, it can generally be used directly in molecular biology applications, or it can be further purified. The purification of fully deprotected DNA is more tedious than the purification of DMT-DNA. The purification of fully deprotected DNA is usually done by ion exchange chromatography or by gel purification. Such operations, including final evaporation, often take at least one full day to perform. Using modern automated DNA synthesizers and high quality reagents, synthetic DNA is typically not purified, particularly when recovered in the fully deprotected state. The DMT-DNA is more suitable for rapid purification, in methods where the lipophilic DMT group can be used in affinity purification. The purification scheme on a cartridge takes about 20 minutes and the final evaporation about 2 hours, before the DNA is ready to be used.
As an alternative to evaporation, precipitation of naturally-occurring DNA has been used for purification purposes. For synthetic DNA, however, precipitation is not typically used as a recovery means, although the possibility has been recently introduced. A product identified as "Oligoclean.TM." has been introduced by United States Biochemical Corporation, Cleveland, Ohio. This product appears to contain alcohol and acetic acid.
The manufacturer's recommended procedure for oligonucleotide purification using the Oligoclean.TM. product involves the addition of 1 ml of that product to 0.1 ml of concentrated ammonium hydroxide. This is followed by incubation at -20.degree. C. for 45 minutes, or overnight for shorter oligonucleotides or those high in A-T content.
It would appear that the Oligoclean.TM. product would not be particularly useful for the recovery of synthetic DNA, since considerably more ammonium hydroxide is typically used in traditional ammonium hydroxide cleavage protocols (e.g., on the order of 3 ml). Using 3 ml of ammonium hydroxide, for instance, the Oligoclean.TM. procedure recommended by the manufacturer would need to be repeated about 30 times in order to recover the entire sample.
One reason that ammonium-based cleavage procedures have not been traditionally followed by recovery by precipitation may be related to the increased solubility of the resultant DNA ammonium, as compared to that of a DNA sodium salt.
In view of the foregoing, it appears that conventional protocols for the cleavage, deprotection, and recovery of synthetic DNA are typically ammonia-based protocols, and commonly require on the order of 10 to 15 hours to complete. As seen by the above-described attempt by Beckman, there is an understanding that it would advantageous to be able to shorten the time required for cleavage/deprotection and recovery of synthetic DNA to on the order of one hour or less. Shorter processing times would provide significant advantages in terms of safety, speed, labor reduction, and ease of automation. What is clearly needed are reagents for the rapid cleavage/deprotection and recovery (neutralization/precipitation) of the entire samples of newly synthesized oligonucleotides, particularly those prepared via the phosphotriester and phosphoramidite synthetic route.
Co-pending international patent application Ser. No. PCT/US93/03123 describes and claims an apparatus and related process for performing automated or semi-automated operations in the course of the removal, recovery, deprotection, and/or purification (recovery) of biopolymer attached to a solid matrix that is contained in a reaction chamber commonly shaped as a column or cartridge. The apparatus allows the delivery of liquids containing chemicals and/or solvents into one end of the column, and the collection (i.e., recovery) of liquids at the opposite end of the column, e.g., for the collection of product. Preferred reagents are described as including a basic reagent for the cleavage deprotection step followed by a neutralizing reagent for precipitation of the oligomer.